Smac Mimetic Research Articles

Smac mimetics as novel promising modulators of apoptosis in the treatment of breast cancer.

Breast cancer is the most prevalent cancer in women. Despite improvements in treatment, the rate of breast cancer-related deaths is still high, and this issue needs further, accurate investigations. Although several treatment options are available, none of them are efficient for complete remission, particularly in advanced stages of the disease. It is known that cancerous cells have dysregulated apoptosis-related pathways, by which they can remain alive for a long time, expand freely, and escape from apoptosis-inducing drugs or antitumor immune responses. Therefore, modulation of apoptosis resistance in cancer cells may be an efficient strategy to overcome current problems faced in the development of immunotherapeutic approaches for the treatment of breast cancer. The inhibitors of apoptosis protein (IAPs) are important targets for cancer therapy because it has been shown that these molecules are overexpressed and highly active in various cancer cells and suppress apoptosis process in malignant cells by blockage of caspase proteins. There is evidence of Smac mimetics efficacy as a single agent; however, recent studies have indicated the efficacy of current anticancer immunotherapeutic approaches when combined with Smac mimetics, which are potent inhibitors of IAPs and synthesized mimicking Smac/Diablo molecules. In this review, we are going to discuss the efficacy of treatment of breast cancer by Smac mimetics alone or in combination with other therapeutics.

DEBIO 1143, an IAP inhibitor, reverses carboplatin resistance in ovarian cancer cells and triggers apoptotic or necroptotic cell death

The poor prognosis of ovarian cancer (it is the leading cause of death from gynecological cancers) is mainly due to the acquisition of resistance to carboplatin. Among the possible resistance pathways, resistance to apoptosis and especially the overexpression of inhibitor of apoptosis proteins (IAP) cIAP1 and X-linked IAP (XIAP), have been implicated. DEBIO 1143, a SMAC (second mitochondria-derived activator of caspase) mimetic, belongs to a new class of targeted agents currently being evaluated in clinical trials, which activate apoptotic cell death and block pro-survival signaling in cancer cells. Here, we demonstrate that DEBIO 1143 in vitro inhibits the cell viability of two carboplatin-sensitive cell lines (IGROV-1 and A2780S) as well as three carboplatin-resistant cell lines (A2780R, SKOV-3 and EFO-21). Of note, DEBIO 1143 is able to reverse resistance to carboplatin by inducing cell death either by apoptosis or necroptosis depending on the cell lines. To identify a biomarker able to predict the sensitivity of the cell lines to DEBIO 1143 treatment we analyzed the expression of the DEBIO 1143 targets cIAP1 and XIAP, and one of their downstream targets, caspase 9. These proteins did not constitute a marker of DEBIO 1143 sensitivity/resistance. Importantly, we confirmed these findings in vivo in SKOV-3 xenograft models where DEBIO 1143 highly potentiated carboplatin treatment.

A Single Mouse Trial Platform for Evaluation of Novel Agents in Acute Lymphoblastic Leukemia By the Pediatric Preclinical Testing Consortium

Introduction: While children diagnosed with the most common pediatric malignancy, acute lymphoblastic leukemia (ALL), now experience close to a 90% likelihood of cure, the outcome for several high-risk subtypes remains poor. Furthermore, since standard-of-care drugs are extremely effective in this disease, and there are relatively few patients eligible for early phase clinical trials, only the most promising new agents are advanced for clinical evaluation following rigorous preclinical testing. However, conventional preclinical testing of novel agents is not sufficiently resourced to be able to encompass the vast heterogeneity of pediatric ALL, and new approaches to preclinical testing are required in this disease. The purpose of this study was to evaluate the utility of a single mouse trial (SMT) platform for preclinical assessment of novel agents on an almost clinical trial scale, to encompass the broad heterogeneity of pediatric ALL in a single experiment, and to identify molecular biomarkers associated with in vivo drug responses when carried out using molecularly-annotated patient-derived xenografts (PDXs).Methods: Eighty pediatric ALL PDXs broadly representative of all pediatric ALL subtypes were characterized in terms of engraftment kinetics in immune-deficient NSG mice, and molecularly annotated by RNA-seq, exome-seq and DNA copy number analysis. Between 2-5 million cells from each PDX were inoculated via the tail vein into 2 NSG mice/PDX. Starting at 2 weeks post inoculation engraftment was monitored by flow cytometric enumeration of the proportion of human CD45+cells in the murine peripheral blood (%huCD45+). When the %huCD45+ for each PDX reached >1% one mouse/PDX was treated with the established drug and topoisomerase I inhibitor topotecan (Tpt, 0.6 mg/kg IP daily x 5 x 2 weeks, repeated at 21 days) and the other mouse was treated with the experimental drug and second mitochondria-derived activator of caspases (SMAC)-mimetic birinapant (Bpt, 15 mg/kg IP every 3 days x 5). Treatment response was monitored using stringent objective response criteria modeled after the clinical setting, by mouse event-free survival (EFS where an event was defined as 25% huCD45+), and by waterfall plots comparing the maximum decrease in %huCD45+at any point post treatment initiation. The authenticity of each PDX was verified using a 60-allele SNP array both at the time of inoculation and at relapse post drug treatment for all mice.Results: Retrospective analysis of 1,000 samples of “single mouse” data previously showed that the single mouse results predicted the overall group response from conventional testing 75.3% of the time, which increased to 94.3% if a deviation of ± one response category was allowed. SMT results were achieved for 71 (88.8%) and 73 (91.3%) of the intended 80 mice for Tpt and Bpt, respectively. Waterfall plots revealed that 60/71 (84.5%) and 30/73 (41.1%) of PDXs achieved regressions in response to Tpt and Bpt treatment, respectively. When compared with historical objective response measures from conventional drug testing carried out by the Pediatric Preclinical Testing Consortium (PPTC) the SMT results showed high concordance for both Tpt (R=0.904; P=0.014; n=7 PDXs) and Bpt (R=0.804; P<0.0001; n=20 PDXs). Moreover, the SMT confirmed the preferential in vivo efficacy of Bpt against B-ALL compared with T-ALL, early T-cell precursor ALL (ETP-ALL) and ALL with mixed linage leukemia gene rearrangements (MLLr-ALL) that was previously revealed by conventional testing carried out by the PPTC. Analysis of divergent responses observed within the MLLr-ALL subpanel (n=9 PDXs) to Tpt revealed a potential 40 gene signature model, with up-regulated genes in Responders being associated with transcription regulation, cellular proliferation and differentiation.Conclusions: This study has shown that SMTs provide an accurate and cost-effective platform for preclinical drug testing in pediatric ALL on an almost clinical trial scale. Moreover, SMTs can almost encompass the heterogeneity of pediatric ALL in a single experiment, and are likely to be useful for large-scale correlations with in vitro drug sensitivity data. Finally, SMTs have the power to identify molecular biomarkers of in vivo response to established and novel drugs in pediatric ALL when combined with exome-seq, RNA-seq and DNA copy number analysis of molecularly-annotated PDXs. Supported by U01CA199000 from the NCI. DisclosuresNo relevant conflicts of interest to declare.

LCL161, an Oral Smac Mimetic/IAP Antagonist for Patients with Myelofibrosis (MF): Novel Translational Findings Among Long-Term Responders in a Phase 2 Clinical Trial

LCL161, an Oral Smac Mimetic/IAP Antagonist for Patients with Myelofibrosis (MF): Novel Translational Findings Among Long-Term Responders in a Phase 2 Clinical Trial

The Role of NRAS G12D Mutations in the Response to Conventional Chemotherapy and 5-Azacitidine in Secondary AML

Secondary Acute Myeloid Leukemia (sAML) accounts for 10-30% of all AML. It arises from a preexisting clonal disorder of hematopoiesis, such as myelodysplastic syndromes (MDS) or chronic myeloproliferative neoplasia (cMPN) in most cases (60-70%) or from exposure to a leukemogenic agent e.g. chemotherapy. sAML is generally considered to be of unfavorable prognosis, as treatment sensitivity is reduced, compared to de novo AML (dnAML) and overall survival is shortened. The incidence of AML associated NRAS are similar between sAML and dnAML (10 to 15%, Jelena D. Milosevic et al.). Prognostic impact of such mutations have been controversially discussed, but have been linked to favorable response to high dose cytarabine treatment in dnAML patients (Andreas Neubauer et al.), thus providing the first example of an oncogenic mutation impacting drug response in dnAML. This effect, however, has not yet been shown in sAML, therefore the aim of this work is to study the role of mutated NRAS in the response to chemotherapy and the hypomethylating agent (HMA) 5-azacitidine in sAML.We utilized two sAML cell lines SET-2 and HEL (both NRAS wildtype) in which we stably introduced the NRAS WT and the known activating hotspot mutation NRAS G12D using the sleeping Beauty technology. The dose-response assays of conventional chemotherapy and 5-azacitidine were carried out in the parental cell lines (SET-2/HEL) compared to NRAS WT (SET-2 NRAS WT/HEL NRAS WT) and NRAS G12D (SET-2 NRAS G12D/HEL NRAS G12D). In contrast to our expectations, both NRAS G12D mutation harboring cell lines, SET-2 and HEL developed resistance to cytarabine, idarubicin and 5-azacytidine, whereas the ones with wildtype NRAS remained susceptible to the drugs. To reverse the resistance we tested the MEK inhibitors Binimetinib and Trametinib in our SET-2 NRAS G12D cell line model according to recent reports about preclinical efficacy of MEK inhibition in NRAS mutant dnAML cells (Michael R. Burgess et al.). And in fact, single agent Binetimib and Trametinib treatments reduced cell viability by 20% at 48 hours. Strikingly, in combination with 5-azacitidine, Binimetinib and Trametinib treatments led to a viability reduction by 90%. Next we induced necroptosis in our NRAS mutant cell line models through the combination of Birinapant (SMAC mimetics) and Emricasan (Inhibitor of Caspase 8), as recently described by Brumatti et al. and were, in addition, able to reduce the cell viability by 60 %.In summary, we provide first evidence, that in contrast to dnAML, activating NRAS mutations may promote resistance to conventional chemotherapy and 5-azacitidine in sAML cell lines. Furthermore we were able to demonstrate, that the combination of MEK inhibitors (Binimetinib and Trametinib) and 5-azacitidine as well as the induction of necroptosis such as the combination birinapant and emricasan, may provide a potential strategy to overcome the resistance. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The SMAC Mimetic APG-1387 Sensitizes Immune-Mediated Cell Apoptosis in Hepatocellular Carcinoma.

The inhibitor of apoptosis protein (IAP) genes are frequently overexpressed in malignancies. Second mitochondria-derived activator of caspase (SMAC) mimetics, which target IAPs, have potential to trigger cancer cell death and sensitize tumor cells to cytotoxic therapy. The aim of this study was to investigate the anti-tumor potential of a novel bivalent SMAC mimetic, APG-1387, in hepatocellular carcinoma (HCC). The mRNA and protein expressions of IAPs, including cellular IAPs (cIAP1 and cIAP2) and X chromosome-linked IAP (XIAP), were increased in HCC tumors compared with normal liver tissue. APG-1387 treatment alone significantly reduced the protein levels of IAPs, but had only a modest effect on the viability and apoptosis of HCC cells in vitro. However, APG-1387 in combination with tumor necrosis factor-alpha (TNF-α) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) significantly reduced cell viability and proliferation, and induced apoptosis in HepG2 cells, as well as in HCCLM3 cells that harbors cancer stem cell-like properties. These synergistic killing effects were caspase-dependent and partially dependent on RIPK1 kinase activity. Furthermore, APG-1387 also promoted the killing effect of Natural Killer cells on HCC cells in vitro and the combination therapy significantly inhibited tumor growth by inducing cell apoptosis in xenograft mice model. In conclusion, our study clarified that APG-1387 could sensitize HCC cells to cytokines or immune cells mediated cell killing and implied that potential of SMAC mimetic based combination immunotherapy for HCC treatment.

IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri

Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 μM H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.

E2F1 binds to the peptide-binding groove within the BIR3 domain of cIAP1 and requires cIAP1 for chromatin binding.

The cellular inhibitor of apoptosis 1 (cIAP1) is an E3-ubiquitin ligase that regulates cell signaling pathways involved in fundamental cellular processes including cell death, cell proliferation, cell differentiation and inflammation. It recruits ubiquitination substrates thanks to the presence of three baculoviral IAP repeat (BIR) domains at its N-terminal extremity. We previously demonstrated that cIAP1 promoted the ubiquitination of the E2 factor 1 (E2F1) transcription factor. Moreover, we showed that cIAP1 was required for E2F1 stabilization during the S phase of cell cycle and in response to DNA damage. Here, we report that E2F1 binds within the cIAP1 BIR3 domain. The BIR3 contains a surface hydrophobic groove that specifically anchors a conserved IAP binding motif (IBM) found in a number of intracellular proteins including Smac. The Smac N-7 peptide that includes the IBM, as well as a Smac mimetic, competed with E2F1 for interaction with cIAP1 demonstrating the importance of the BIR surface hydrophobic groove. We demonstrated that the first alpha-helix of BIR3 was required for E2F1 binding, as well as for the binding of Smac and Smac mimetics. Overexpression of cIAP1 modified the ubiquitination profile of E2F1, increasing the ratio of E2F1 conjugated with K11- and K63-linked ubiquitin chains, and decreasing the proportion of E2F1 modified by K48-linked ubiquitin chains. ChIP-seq analysis demonstrated that cIAP1 was required for the recruitment of E2F1 onto chromatin. Lastly, we identified an E2F-binding site on the cIAP1-encoding birc2 gene promoter, suggesting a retro-control regulation loop.

IAP Proteins Antagonist: An Introduction and Chemistry of Smac Mimetics under Clinical Development.

Smac mimetics (also known as IAP antagonist) are a new class of targeted drugs having a goal to suppress the IAPs, reestablishing the apoptotic pathways and inducing cancer cell death. Therefore, development of Smac mimetics was considered an attractive strategy for the development of new anticancer drugs. Lots of reviews have come in yesteryears which mainly discussed the biology of IAPs and their role in cancer development. None of these reviews focused on the chemical synthesis of Smac mimetics. Literature study was done by using standard bibliographic search engines like scifinder, pubmed etc. The characteristic features of screened articles were described in the review. The review gives an introduction of IAP proteins and Smac mimetics. Readers will gain an overview of the development of Smac mimetics with representative examples of both monovalent and bivalent Smac mimetics as anticancer agents and an understanding of their structure-activity relationships. Chemical synthesis of biologically important Smac mimetics was discussed briefly in this review. Small molecules that mimic Smac are continuously progressing towards clinical development. Smac mimetics are generally well tolerated and have demonstrated rapid suppression of their target (the IAPs), activation of apoptosis and anti-tumor activity. Continuous research has been done to generate even more insight into the function of IAP proteins to significantly enhance the therapeutical potential of Smac mimetics.

The modulation of Smac/DIABLO on mitochondrial apoptosis induced by LPS in Crassostrea gigas

The mitochondrial pathway of apoptosis is well studied as the major mechanism of physiological cell death in vertebrates. In the present study, a second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis-binding protein (IAP) with low pI protein (DIABLO) (designated as CgSmac) was identified from oyster Crassostrea gigas. The open reading frame of CgSmac was of 966 bp nucleotides encoding a predicted polypeptide of 321 amino acids with a conserved Smac/DIABLO domain containing a potential IAP-binding motif of VMPV. CgSmac proteins were distributed in hemocytes and co-localized with mitochondria. Western blotting analysis revealed that CgSmac proteins mainly existed in the dimer form in hemocytes, and the monomeric precursors and mature monomers were also detected. After lipopolysaccharide (LPS) stimulation, the mRNA expression of CgSmac in hemocytes was significantly up-regulated and peaked at 6 h (12.26-fold, p < 0.05), and the protein level of its dimers was significantly up-regulated at 6 h, 12 h, 24 h, and 48 h, while that of CgSmac monomers was up-regulated at 6 h, 12 h and down-regulated at 24 h, 48 h. The decrease of mitochondrial membrane potential indicated that the occurrence of early stage of apoptosis in primary cultured hemocytes was induced by LPS, and RNA interference (RNAi) of CgSmac could not rescue this decrease. The caspase-3 activity in primary cultured hemocytes was significantly suppressed after RNAi of CgSmac. Correspondingly, the total apoptotic rate of primary cultured hemocytes was also significantly suppressed in dsCgSmac + LPS group (31.57%) compared to dsEGFP + LPS group (40.27%, p < 0.05), which in turn demonstrated the conserved pro-apoptotic function of CgSmac. Furthermore, the early apoptotic rate (10.4% vs. 8.5%, p < 0.05) was significantly higher in dsCgSmac + LPS group than that of dsEGFP + LPS group, while the necrosis (7.7% vs. 10.0%, p < 0.05) and late apoptotic rates (13.4% vs. 21.9%, p < 0.05) were lower in dsCgSmac + LPS group than those of dsEGFP + LPS group. Collectively, CgSmac could activate mitochondrial apoptosis pathway by promoting caspase-3 activity in oyster hemocytes against exogenous LPS invasion. These results provided new insights on oyster apoptosis and the immune defense mechanisms in invertebrates.

Identification of Smac mimetics as novel substrates for p-glycoprotein

Multidrug resistance (MDR) in cancer patients undergoing chemotherapy is preventing effective treatment of multiple cancer types including pediatric tumors. Resistance to chemotherapeutic drugs in cancer cells is frequently associated with high expression of p-glycoprotein, a transporter in the plasma membrane that can mediate cellular drug export. Here, we generated pediatric cancer cells with acquired resistance to the chemotherapeutic drug vincristine (VCR). In these cells, acquired resistance is associated with increased expression of p-glycoprotein. VCR-resistant cells display an MDR phenotype and have acquired resistance to multiple other chemotherapeutic drugs including doxorubicin (DOXO) and etoposide (ETO). Notably, we discovered that these cells also display cross-resistance with several Smac mimetics, a novel class of experimental cancer therapeutics designed to induce apoptosis by inhibiting Inhibitor of Apoptosis (IAP) proteins. Resistance to Smac mimetics is reversible in the presence of p-glycoprotein inhibitors, highlighting Smac mimetics as novel substrates for p-glycoprotein. The identification of Smac mimetics as substrates for p-glycoproteins may influence the design of future clinical trials to prevent usage of Smac mimetics in the context of MDR or, alternatively, combine Smac mimetics with p-glycoprotein inhibitors to maximize their efficiency.

Application of Native ESI-MS to Characterize Interactions between Compounds Derived from Fragment-Based Discovery Campaigns and Two Pharmaceutically Relevant Proteins

Native electrospray ionization mass spectrometry (ESI-MS) was applied to analyze the binding of compounds generated during fragment-based drug discovery (FBDD) campaigns against two functionally distinct proteins, the X-linked inhibitor of apoptosis protein (XIAP) and cyclin-dependent kinase 2 (CDK2). Compounds of different molecular weights and a wide range of binding affinities obtained from the hits to leads and lead optimization stages of FBDD campaigns were studied, and their dissociation constants (Kd) were measured by native ESI-MS. We demonstrate that native ESI-MS has the potential to be applied to the stages of an FBDD campaign downstream of primary screening for the detection and quantification of protein–ligand binding. Native ESI-MS was used to derive Kd values for compounds binding to XIAP, and the dissociation of the complex between XIAP and a peptide derived from the second mitochondria-derived activator of caspases (SMAC) protein induced by one of the test compounds was also investigated. Affinities of compounds binding to CDK2 gave Kd values in the low nanomolar to low millimolar range, and Kd values generated by MS and isothermal titration calorimetry (ITC) followed the same trend for both proteins. Practical considerations for the application of native ESI-MS are discussed in detail.

Combination of a Latency-Reversing Agent With a Smac Mimetic Minimizes Secondary HIV-1 Infection in vitro.

Latency-reversing agents (LRAs) are considered a potential tool to cure human immunodeficiency virus type 1 (HIV-1) infection, but when they are taken alone, virus production by reactivated cells and subsequent infection will occur. Hence, it is crucial to simultaneously take appropriate measures to prevent such secondary HIV-1 infection. In this regard, a strategy to minimize the production of infectious viruses from LRA-reactivated cells is worth pursuing. Here, we focused on a second mitochondria-derived activator of caspases (Smac) mimetic, birinapant, to induce apoptosis in latent HIV-1-infected cells. When birinapant was administered alone, it only slightly increased the expression of caspase-3. However, in combination with an LRA (e.g., PEP005), it strongly induced the expression of caspase-3 followed by enhanced apoptosis. Importantly, the combination eliminated reactivated cells and drastically reduced HIV-1 production. Finally, we found that birinapant decreased the mRNA expression of HIV-1 that was induced by PEP005 in the primary CD4+ T-cells from HIV-1-carrying patients as well. These results suggest that the combination of an LRA and an “apoptosis-inducing” agent, such as a Smac mimetic, is a possible treatment option to decrease HIV-1 reservoirs without the occurrence of HIV-1 production by reactivated cells.

Inhibition of cIAP1 as a strategy for targeting c-MYC–driven oncogenic activity

Protooncogene c-MYC, a master transcription factor, is a major driver of human tumorigenesis. Development of pharmacological agents for inhibiting c-MYC as an anticancer therapy has been a longstanding but elusive goal in the cancer field. E3 ubiquitin ligase cIAP1 has been shown to mediate the activation of c-MYC by destabilizing MAD1, a key antagonist of c-MYC. Here we developed a high-throughput assay for cIAP1 ubiquitination and identified D19, a small-molecule inhibitor of E3 ligase activity of cIAP1. We show that D19 binds to the RING domain of cIAP1 and inhibits the E3 ligase activity of cIAP1 by interfering with the dynamics of its interaction with E2. Blocking cIAP1 with D19 antagonizes c-MYC by stabilizing MAD1 protein in cells. Furthermore, we show that D19 and an improved analog (D19-14) promote c-MYC degradation and inhibit the oncogenic function of c-MYC in cells and xenograft animal models. In contrast, we show that activating E3 ubiquitin ligase activity of cIAP1 by Smac mimetics destabilizes MAD1, the antagonist of MYC, and increases the protein levels of c-MYC. Our study provides an interesting example using chemical biological approaches for determining distinct biological consequences from inhibiting vs. activating an E3 ubiquitin ligase and suggests a potential broad therapeutic strategy for targeting c-MYC in cancer treatment by pharmacologically modulating cIAP1 E3 ligase activity.

Structure-based design, synthesis and anticancer effect of cyclic Smac-polyarginine peptides.

The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mitochondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring field for cancer therapy. In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly showed that cyclic peptides P4, P5 and P7 gave rise to a significant level of cytotoxicity and apoptosis mediated cell death in multiple myeloma tumor cells (MM) comparing to linear peptide.

Synthesis and anticancer activity of conformationally constrained Smac mimetics containing pseudo β turns

Herein, we report synthesis and in vitro anticancer activity of conformationally constrained Smac mimetics containing reverse turn inducing motifs “Ant-Pro” and “sAnt-Pro”. The synthesis of Smac analogs with diverse hydrophobic groups at the C-terminus was carried out using solution phase peptide synthesis. The synthesis of Ant-Pro containing analogs 3a–j was carried out by ring opening of benzoxazinones 7a–c, whereas, their sulfonamide counterparts 4a–h were synthesized by using routine acid-amine coupling reaction. In vitro anticancer studies against breast cancer cell line MDA-MB-231 revealed that some of the new analogs had better anticancer activity than the standard AVPI Smac tetrapeptide.

Abstract 1754: Smac mimetics APG-1387 synergizes with immune checkpoint inhibitors in preclinical models

Abstract APG-1387 is a novel, SMAC-mimetic, small molecule inhibitor for inhibitor of apoptosis proteins (IAPs). IAPs are a class of proteins which negatively regulate caspases and apoptosis. The IAP proteins are frequently dysregulated in many cancers and have been suggested as contributing to apoptosis-resistance of cancer cells to a variety of anticancer drugs. DNA amplification of the cellular IAP-1 (cIAP-1) and cellular IAP-2 (cIAP-2) genes (BIRC2 and BIRC3, respectively) has been observed in various human cancer types, including lung cancer, pancreatic cancer, and liver carcinoma. Dysregulation of IAP proteins has also been frequently observed at the protein level in multiple cancer cell lines and tumor samples. IAPs contribute to tumor cell survival, chemo-resistance, disease progression, and poor prognosis. In addition, IAPs play an important role in immune regulation. For example, IAPs regulate ubiquitin (Ub)-dependent pathways that modulate innate immune signaling via activation of nuclear factor κB (NF-κB). Due to their significant biological functions in cell death and immune responses, IAPs have been emerging as drug targets in a wide range of malignancies. Several IAP inhibitors have been developed and their clinical benefits are currently in investigation in multiple clinical trials in both single agent and combination regimen with immune checkpoint inhibitors. APG-1387 has been developed as a novel drug candidate targeting IAPs. Mechanistically, in both cancer cells and xenograft tumors, APG-1387 induces degradation of cIAP-1 and XIAP proteins, as well as caspase-3 activation and PARP cleavage, which leads to apoptosis. Anti-proliferative and anti-tumor activities of APG-1387 have been well demonstrated in a panel of cancer cell lines and xenograft tumor models. In this study, we explored the synergistic effect of APG-1387 in combination therapy with immune checkpoint inhibitors. We found that APG-1387 enhanced human PBMC co-stimulation of anti-CD3 and anti-CD28, and T cell proliferation in vitro. In syngeneic tumor tissues, the combination treatment with APG-1387 and anti-PD-1 antibody led to an increase in CD3+ and CD8+ T cell populations and a decrease in Treg cell population. Synergistic effect of the combination was further demonstrated in syngeneic tumor models. Taken together, our results suggest that the treatment with APG-1387 may enhance the effect of anti-PD-1 antibody therapy. Consequently, APG-1387 has been approved by FDA for phase I clinical trials as single agent and in combination with anti-PD-1 antibody in solid tumors. Citation Format: Wentao Pan, Douglas D. Fang, Guangfeng Wang, Miaoyi Wu, Yingfeng Li, Keji Lian, Dingxiong Cheng, Yanhui Kong, Guoqin Zhai, Ming Guo, Shaomeng Wang, Dajun Yang, Yifan Zhai. Smac mimetics APG-1387 synergizes with immune checkpoint inhibitors in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1754.

Abstract 4399: Entinostat sensitizes colorectal cancer cell lines to the IAP antagonist, ASTX660 by down-regulating FLIP expression

Abstract Introduction Inhibitor of Apoptosis Proteins (IAPs) are anti-apoptotic proteins which suppress the activation of caspases. IAPs have been shown to be overexpressed in several cancers, including colorectal cancer (CRC) and correlate with poor prognosis. IAP antagonists, including SMAC mimetics, can convert TNFα from a pro-inflammatory and pro-survival signal into a potent pro-death signalling molecule. In the presence of IAP antagonists, TNFα signalling is driven to cell death induction through the formation of a complex containing FADD/procaspase 8 and RIPK1, termed complex-II. However, high expression of FLIP, an endogenous regulator of procaspase-8 processing and activation, can inhibit cell death signalling from this complex. We have previously shown that FLIP is frequently overexpressed in CRC, and we and others have demonstrated that histone deacetylase (HDAC) inhibition results in down-regulation of FLIP protein expression. The aims of this study were to assess the efficacy of a novel, non-peptidomimetic, cIAP and XIAP antagonist, ASTX660 (Astex Pharmaceuticals), as single agent and in combination with the class I HDAC inhibitor Entinostat in CRC cell lines. Results ASTX660 was found to be a potent on-target IAP antagonist in a panel of human and murine CRC cell lines, resulting in reduced cIAP1 expression and formation of the apoptosis-inducing complex II, containing RIPK1 and caspase-8, in the presence of TNFα. However, viability of CRC cell lines was unaffected by the ASTX660 treatment alone or in the presence of TNFα, added to mimic the pro-inflammatory microenvironment frequently associated with CRC. Downregulation of FLIP expression using siRNA in a panel of CRC cell lines significantly enhanced cell death induced by ASTX660 in the presence of TNFα. Entinostat down-regulated FLIP protein expression at clinically achievable concentrations and potentiated ASTX660-mediated cell death in the panel of CRC cell lines. This cell death was confirmed to be apoptotic in nature and, more specifically, caspase-8-dependent using CRISPR-caspase-8 knockout models. Moreover, the mode of combination effects was demonstrated to be FLIP-dependent using CRC models engineered to overexpress wild-type FLIP. Conclusion High levels of FLIP expression in pre-clinical models of CRC may contribute to resistance to single agent IAP antagonist treatment. Pre-treatment with the class I HDAC inhibitor Entinostat can sensitize CRC cell lines toASTX660 by down-regulating FLIP expression. HDAC inhibitors could be used in novel combinations with IAP antagonists to promote apoptosis in pro-inflammatory CRC. Citation Format: Nyree T. Crawford, Katie Stott, Tomoko Smyth, John Lyons, Roberta Ferraldeschi, George Ward, Daniel B. Longley. Entinostat sensitizes colorectal cancer cell lines to the IAP antagonist, ASTX660 by down-regulating FLIP expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4399.

Interferon-\u03b3 and Smac mimetics synergize to induce apoptosis of lung cancer cells in a TNF\u03b1-independent manner

BackgroundThe prognosis of lung cancer is very poor and hence new therapeutic strategies are urgently desired. In this study, we searched for efficacious Smac mimetic-based combination therapies with biomarkers to predict responses for non-small cell lung cancer (NSCLC).MethodsNSCLC cell lines and normal human alveolar epithelial cells were treated with Smac mimetics plus IFNγ or other agonists and cell viabilities were assessed by MTS assay, cell counting, flow cytometry and cell colony assay. Western blot analysis was performed to assess the cleavage (activation) of caspases and expression of signaling molecules. Caspase activity was determined to verify caspase activation. The pathways involved in NSCLC cell death were investigated using specific inhibitors.ResultsWe found that IFNγ could cooperate with various Smac mimetics to trigger a profound apoptosis in a number of NSCLC cell lines that are competent for IFNγ signaling (i.e. expressing IFNγ receptor-1 and STAT1) but have low expression levels of inhibitor of apoptosis proteins survivin and livin without harming normal human lung epithelial cells. IFNγ co-treatment with a novel class dimeric Smac mimetic AZD5582 eradicated NSCLC cell colony formation. Unlike IFNγ, IFNα, IFNλ, TNFα, or TRAIL alone or plus AZD5582 had minor effects on NSCLC cell viability. IFNγ/AZD5582-induced cell death in NSCLC cells was independent of TNFα autocrine but relied on apoptosis mediated by JAK kinase, caspase 8 and RIPK1 pathways.ConclusionOur results indicate that IFNγ and Smac mimetics can synergize to induce apoptosis of NSCLC cells and suggest that IFNγ and Smac mimetic regimen may be a novel and efficacious apoptosis targeted therapy with biomarkers to predict responses for NSCLC cells.

Resveratrol Sensitizes Carfilzomib-Induced Apoptosis via Promoting Oxidative Stress in Multiple Myeloma Cells.

The proteasome inhibitor is a target therapy for multiple myeloma (MM) patients, which has increased the overall survival rate of multiple myeloma in clinic. However, relapse and toxicity are major challenges for almost all MM patients. Thus, there is an urgent need for an effective and less toxic combination therapy. Here, we demonstrated that a natural compound, resveratrol (RSV) displayed anti-proliferative activity in a dose- and time-dependent manner in a panel of MM cell lines. More importantly, a low concentration of RSV was synergistic with a low dose of the proteasome inhibitor carfilzomib (CFZ) to induce apoptosis in myeloma cells. Further studies showed that mitochondria was a key regulatory site after RSV/CFZ combination treatment. RSV induced the release of second mitochondria-derived activator of caspase (Smac) in a dose-dependent manner and kept the Smac in a high level after combination with CFZ. Also, RSV was additive with CFZ to increase reactive oxygen species (ROS) production. Moreover, a stress sensor SIRT1, with deacetylase enzyme activity, was remarkably downregulated after RSV/CFZ combination, thereby significantly decreasing its target protein, survivin in MM cells. Simultaneously, autophagy was invoked after RSV/CFZ combination treatment in myeloma cells. Further inhibition of autophagy could increase more ROS production and apoptosis, indicating a close linkage between autophagy and proteasome to modulate the oxidative stress. Together, these findings suggest that induction of multiple stress responses after RSV/CFZ combination is a major mechanism to synergistically inhibit MM cell growth and reduce the toxicity of CFZ in MM cells. This study also provides an important rationale for the clinic to consider an autophagy inhibitor for the combination therapy in MM patients.